The present invention relates to an apparatus for carrying out chromatographic separations by means of the overpressured thin-layer chromatography. According to art the known apparata include a carrier plate covered by a sorbent layer receiving the samples to be investigated and elements defining a closed space wherein overpressure can be generated for pressing a flexible element to the sorbent layer or the sorbent layer to an appropriate element. The efficiency of the separation technique realised by means of overpressure thin-layer chromatography can be improved when applying the invented apparatus.
The advantages offered by the column chromatography and its planar version, the so called thin-layer chromatography, can be effectively combined by the method of overpressured thin-layer chromatography presented e.g. in GB-PS 1 570 760. According to the specification of this patent an apparatus for realizing the method includes an overpressured chamber adjacent to a sorbent layer. As sorbent the same organic and inorganic materials can be applied as in both parent methods. The sorbent layer is covered completely by a plastic plate made advantageously of flexible material which is pressed to the layer by means of overpressure generated in the closed space, e.g. by water supplied therein. Means are applied for blocking the solvent from leaving the sorbent layer, e.g. by a sealing layer around this layer to be connected with the plastic plate. This results in virtual elimination of the vapor space which is characteristic for the known thin-layer chromatography. The vapor phase realized over the sorbent layer is deemed to be the source of some disadvantages. The plate carrying the sorbent layer should be, as mentioned, sealed on the edges in order to prevent leaking of the eluant (solvent) admitted to the sorbent layer under the influence of overpressure transmitted by the plastic plate. The migration of the eluant can be prevented by means of channels formed in the sorbent layer or by a plastic plate pressed onto the surface of this layer.
For improving the efficiency of the known overpressured thin-layer chromatographic techniques a higher number of sorbent layers can be arranged in a chamber. In this case the carrier plates are stacked above one another and form a parallel system wherein the sorbent layers lie on the same side of these plates. An apparatus of this kind is shown e.g. in Hungarian Patent Application 1335/82, the early publication of which took place in February 1985. The carrier plates should be sealed to one another on their edges by applying an appropriate sealing medium. The overpressured closed space can be realized either over the upper or under the lower carrier plate and in this case the carrier plates transmit the overpressure in the system. Usually the carrier plates are equipped with a system of parallel channels beginning from one of the edges or from the middle of the sorbent layer, wherein the eluant can communicate between the sorbent layers.
The chromatographic separation cannot be carried out with high efficiency unless the basic conditions of separation are selected in an appropriate way. The basic conditions of liquid chromatography are: the material of the sorbent, the mobile phase--eluant, the temperature of the separation, etc. The temperature is an important parameter in the gas chromatography techniques and it is selected therein very carefully. The desired temperature value is maintained during the separation with high accuracy. To date the skilled artisans have believed that the role of temperature is not important in liquid chromatography, as it follows clearly from the practice of carrying out column chromatographic techniques.
The known apparata of thin-layer chromatography work practically with taking into account thermal effects. For example, in the book of J. C. Touchstone and M. F. Dobbins, "Practice of Thin-Layer Chromatography", edited by J. Wiley, New-York, 1978 it is stated on p. 304 that the role of temperature is unimportant in this technique. The statement follows obviously from the fact that a vapor space is present over the sorbent layer in the known apparata and this space has a disadvantageous influence on the conditions of the separation at increased temperatures. For detecting small amounts of different materials it has been proposed to use increased temperature around the chamber during separation and this proposal relates to the method of multiple development wherein the increased temperature is applied for vaporization of the solvent used in the previous development which should be expelled. In a single development the increased temperature and the thereby accelerated vaporization of the solvent can cause many problems because of the closed vapor space over the sorbent layer and therefore the separations are often evaluated with low efficiency and reliability. The vapor of the solvent is a source of inaccuracy.
Of course, the sorbent layer can be placed together with the chamber in an incubator ensuring a stable temperature value selected according to the materials to be separated. This is a known feature of the thin-layer chromatographic developments.
In comparison to the traditional gas chromatographic techniques, the classical liquid column chromatography offers, owing to the large sorbent particles applied, decreased speed and efficiency. The liquid column chromatography was developed because the gas chromatography is applicable only to about 20% of all organic substances. The planar methods, and especially the thin-layer chromatography techniques, proved very useful in the view of the following advantages: they are simple and highly economical in time and materials, and offer the possibility of visual detection and simultaneous study of a large number of samples wherein aggressive reagents may be applied, too. Some drawbacks should be mentioned also. These are the limitation of the number of separations by the length of the carrier plate and the long time required for development. The overpressured thin-layer chromatography methods ensure rapid and effective separation of mixtures of various organic and inorganic substances which are effected about 5 to 20 times faster than by the traditional thin-layer methods, however, they require applying smaller and more uniform sorbent particles than was previously the case.
The efficiency of the separation and its rapidity are in many cases not satisfactory, especially in the case of mixtures comprising a large number of organic compounds, wherein the separation is weak. It is difficult to separate the materials and evaluate the chromatograms obtained.
The problem of the invention is to elaborate a method and an apparatus for improving the efficiency and rapidity of the separations carried out by means of the overpressured thin-layer chromatographic methods.
The invention is based on the recognition that the overpressured thin-layer chromatography can be improved in spectacular manner when applying a temperature program during development. The temperature is a very important parameter--in spite of the beliefs holding sway to date in the field of thin-layer chromatograhy--when the vapor phase can be eliminated, i.e. in the methods of the overpressured thin-layer chromatography. It is important also that the eluant flows under the influence of the overpressure in predetermined directions and that the speed of this flow can be altered by the temperature, too. According to the foregoing recognition the basic feature of the invention lies not in the application of the temperature itself, but in the changes thereof. Thereby it is possible to cause changes in the sequence (in the spatial arrangement, of the materials separated in the sorbent layer. This improves the separations of the different components of the mixture in comparison to the known methods. The higher speed of separation ensured by the increased temperature makes it possible to use also on-line methods of evaluation. The invention is based further on the fact that the increased temperature causes the material pressed to the sorbent layer to absorb the eluant on another condition than in the case of lower temperature and therefore a very limited space of special vapor pressure is realized over the sorbent layer, influencing the separation, the order (sequence) of the components and especially the efficiency in advantageous manner.